Reaction mass of 7,7-dimethyl-2-methylidenebicyclo[2.2.1]heptane and (1R)-2,2-dimethyl-3-methylenebicyclo[2.2.1]heptane and (1S)-2,2-dimethyl-3-methylenebicyclo[2.2.1]heptane and (1S)-2,6,6-trimethylbicyclo[3.1.1]hept-2-ene

Endpoint summary

Description of key information

Skin irritation. Key study. Method according to OECD guideline 439, GLP study. The test item is a skin irrtant (category 2) since the mean percent viability of the treated tissues was 2.4% in the RHE test.

Skin corrosion. Key study. Method according to OECD guideline 431, GLP study. Under RHE test method performed in SkinEthic RHE® model the test item does not have to be classified as skin corrosive (cat. 1).

Eye irritation. Key study. Method according to OECD guideline 437, GLP study. The test item is not irritating to the eye (no category) as the mean IVIS score in the BCOP test was found to be 1.20.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

12 December 2017 - 16 December 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Remarks:

SkinEthic RHE® model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: foreskin (number of donors not specified)

Source strain:

not specified

Justification for test system used:

This study makes use of reconstructed human epidermis (RhE) (obtained from human derived non-transformed epidermal keratinocytes) which closely mimics the histological, morphological, biochemical and physiological properties of the upper parts of the human skin, i.e., the epidermis. Use of reconstructed human epidermis (RhE) is also recommended by OECD and other regulatory authorities. SkinEthicTM RHE model has been validated and is part of OECD validated reference methods (VRMs) and is also a recommended model for conducting in vitro skin irritation studies.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: SkinEthic RHE® model
- Tissue batch number(s): 17-RHE-127
- Production date:
- Shipping date:
- Delivery date:
- Date of initiation of testing: 12/12/2017

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature
- Temperature of post-treatment incubation (if applicable): 37ºC

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: 25 x 1 mL of DPBS
- Observable damage in the tissue due to washing: no
- Modifications to validated SOP: no

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 300 µL of a MTT solution at 1.0 mg/mL
- Incubation time: 3 hours at 37°C, 5% CO2
- Spectrophotometer: Synergy™ microplate reader (BioTek)
- Wavelength: 570 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: 1.4 (CV = 2.8%) specification OD > 0.7. Historical negative control mean OD range = 0.834-1.574.
- Barrier function: 4.8 h (Specification 0h < ET50< 10h)
- Morphology: 5.5 Cell layers, absence of significant histological abnormalities, well differentiated epidermis, specification > 4
- Contamination: no

NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE: no interference.

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be irritant to skin if the tissue viability after 42 minutes exposure is less than or equal to 50%.
- The test substance is considered to be non-irritant to skin if the tissue viability after 42 minutes exposure is greater than 50%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 16 µL (32 µL/cm2)

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 16 µL

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 16 µL
- Concentration (if solution): 5% SDS

Duration of treatment / exposure:

42 minutes

Duration of post-treatment incubation (if applicable):

42 hours ± 60 minutes

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

mean

Value:

2.4

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

(DPBS)

Positive controls validity:

valid

Remarks:

1.2% viability (5% SDS)

Remarks on result:

positive indication of irritation

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system: no
- Direct-MTT reduction: no
- Colour interference with MTT: no

DEMONSTRATION OF TECHNICAL PROFICIENCY: yes. A full demonstration of proficiency was performed for the EpiSkin-SM model, plus a reduced validation with the SkinEthic RHE model. Adequate results were obtained for the evaluated chemicals.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes, SD of the negative control group was 0.03% (acceptablility criteria, SD ≤ 18%) and OD mean is 2.002 (acceptability criteria, 0.8≤OD≤3).
- Acceptance criteria met for positive control: yes, SD of the positive control group was 0.06% (acceptablility criteria, SD ≤ 18%)
- Acceptance criteria met for variability between replicate measurements: yes. SD of test item was 0.06% (acceptablility criteria, SD ≤ 18%).

Table 1: Data Summary of Percent Viability

Treatment	Tissue Replicate	O.D. at 570 nm	Blank Corrected O.D.	Mean of Corrected O.D.	Mean O.D. of Three Tissues	% Viability/ Tissue	Mean % Viability	S.D. of % Viability	C.V. of % Viability	Corrosivity Class
Negative Control (Dulbecco’s Phosphate Buffered Saline (DPBS))	1	2.067	2.026	2.004	2.002	100	100	0.03	1.5	NA
		2.017	1.976
		2.051	2.01
	2	2.021	1.98	1.988
		2.033	1.992
		2.034	1.993
	3	2.019	1.978	2.014
		2.105	2.064
		2.042	2.001
Reaction Mass (Fenchene, Laevo Alpha Pinene, Laevo Camphene, Dextro Camphene)	1	0.092	0.051	0.05	0.049	2.5	2.4	0.06	2.50	Category 2
		0.09	0.049
		0.09	0.049
	2	0.088	0.047	0.048		2.4
		0.089	0.048
		0.089	0.048
	3	0.089	0.048	0.048		2.4
		0.089	0.048
		0.089	0.048
Positive control (Sodium dodecyl sulphate (5% aq.))	1	0.068	0.027	0.026	0.025	1.3	1.2	0.06	5.00	Category 2
		0.067	0.026
		0.066	0.025
	2	0.067	0.026	0.025		1.2
		0.065	0.024
		0.066	0.025
	3	0.066	0.025	0.025		1.2
		0.065	0.024
		0.066	0.025

Key: O.D. = Optical density, S.D. = Standard deviation, C.V. = Coefficient of variation, NA = Not applicable

Note: For Negative control SD and CV of % viability was calculated using OD at 570 nm, and for test item and positive control SD and CV of % viability was calculated using % viability/tissue

Interpretation of results:

other: Skin irritant (Cat. 2) (CLP Regulation EC no. 1272/2008)

Conclusions:

The test substance was found to be a skin irritant (Cat. 2) in a reconstructed human epidermis test.

Executive summary:

An in vitro skin irritation test of the test item was performed in a reconstructed human SkinEthic RHE® model, according to OECD TG 439 (GLP study). Three epidermis units were treated with 16 μL test item for 42 minutes at room temperature. Exposure of the test item was terminated by rinsing with 25 x 1 mL of DPBS. The epidermis units were then incubated at 37°C for 42 hours in an incubator with 5% CO2. The viability of each disk was assessed by incubating the tissues with MTT, extracting the precipitated formazan crystals using isopropanol and incubating the tissues for overnight in a refrigerator protected from light, and finally measuring the concentration of formazan by determining the OD at 570 nm. Under the test conditions, the mean percent viability of the treated tissues was 2.4%, versus 1.2% of the positive control (5% Sodium Dodecyl Sulfate) and 100% of the negative control (DPBS). Therefore, the test item must be considered as classified for skin irritation (Cat. 2).

Reference 2

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

10 March 2018 – 23 March 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Remarks:

SkinEthic RHE® model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: not specified

Justification for test system used:

This study makes use of reconstructed human epidermis (RhE) (obtained from human derived non-transformed epidermal keratinocytes) which closely mimics the histological, morphological, biochemical and physiological properties of the upper parts of the human skin, i.e., the epidermis. Use of reconstructed human epidermis (RhE) is also recommended by OECD and other regulatory authorities. SkinEthicTM RHE model has been validated and is part of OECD validated reference methods (VRMs) and is also a recommended model for conducting in vitro skin irritation studies.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: SkinEthic RHE® model
- Tissue batch number(s): 18-RHE-029
- Production date:
- Shipping date:
- Delivery date:
- Date of initiation of testing: 10/03/2018

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature for 3 min exposure and 37°C ± 1°C for 1 hour exposure.
- Temperature of post-treatment incubation (if applicable): 37ºC

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: 20 x 1 mL of DPBS
- Observable damage in the tissue due to washing: no
- Modifications to validated SOP: no

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1.0 mg/mL
- Incubation time: 3 hours at 37°C, 5% CO2
- Spectrophotometer: Synergy™ microplate reader (BioTek)
- Wavelength: 570 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: OD =1.3 (CV = 6.6%) specification OD > 0.7. Historical negative control mean OD range = 1.610-2.594 (3 min exposure) and 1.806-2.693 (60 min exposure) (acceptability criteria, 0.8≤OD≤3).
- Barrier function: 4.7 h (Specification 4.0h < ET50< 10h)
- Morphology: 6.5 cell layers, absence of significant histological abnormalities, well differentiated epidermis, specification > 4
- Contamination: no

NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE:
- Freeze killed tissues (for positive control)
- Procedure used to prepare the killed tissues (if applicable): Killed tissues were prepared by incubating the viable tissues at -80 ± 5°C for 48 hours.
- N. of replicates : 2
- Method of calculation used: The true tissue viability is calculated as the percent tissue viability obtained with living tissues exposed to the positive minus the percent non-specific MTT reduction obtained with the killed tissues exposed to the same, calculated relative to the negative control run concurrently (%NSMTT). NSMTT was < 0% relative to the negative control, therefore true tissue viability was not determined.

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be corrosive to skin if the viability after 3 minutes exposure is less than 50%, or if the viability after 3 minutes exposure is greater than or equal to 50 % and the viability after 1 hour exposure is less than 15%.
- The test substance is considered to be non-corrosive to skin if the viability after 3 minutes exposure is greater than or equal to 50% and the viability after 1 hour exposure is greater than or equal to 15%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 40 µL (80 µL/cm2)

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 40 µL

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 40 µL
- Concentration (if solution): N/A

Duration of treatment / exposure:

3 minutes and 60 minutes

Duration of post-treatment incubation (if applicable):

3 hours (incubation in MTT solution)

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

mean after 3 min exposure

Value:

84.9

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

(distilled water)

Positive controls validity:

not applicable

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

mean after 1 hour exposure

Value:

26.8

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

(distilled water)

Positive controls validity:

valid

Remarks:

0.1% viability (8N KOH)

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system: no
- Direct-MTT reduction: For adapted positive control treated tissues, NSMTT was < 0% relative to the negative control, therefore true MTT metabolic conversion of treated tissue was not determined.
- Colour interference with MTT: no

DEMONSTRATION OF TECHNICAL PROFICIENCY: yes. A demonstration of proficiency was performed for the SkinEthic RHE® model. Adequate results were obtained for the evaluated chemicals.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes. The mean OD of negative control tissues for treatment time 3 min and 1 hour were 0.974 and 1.037 (acceptability criteria, 0.8≤OD≤3 for the SkinEthic RHE® model).
- Acceptance criteria met for positive control: yes. Mean viability of the tissue replicates exposed for 1 hour, expressed as % of the negative control, is 0.1% < 15%.
- Acceptance criteria met for variability between replicate measurements: yes. 0.9% (for 3 min exposure) and 0.56% (for 60 min exposure) < 30%.

Table 1: Data Summary of Percent Viability

Treatment	Exposure Time	Tissue Replicate	O.D.	Corrected O.D.	Mean of Corrected O.D.	Mean O.D. of Replicate tissues	% Viability/ Tissue	Mean % Viability	SD of % Viability	% CV of % Viability	Corrosivity Class
Negative Control (Sterile Distilled water)	3 Minutes	1	1.015	0.973	0.969	0.974	100	100	0.01	1.0	NA
			0.996	0.954
			1.022	0.980
		2	0.991	0.949	0.983
			1.036	0.994
			1.048	1.006
		3	1.005	0.963	0.971
			1.038	0.996
			0.996	0.954
	60 Minutes	1	0.938	0.896	1.007	1.037	100	100	0.03	2.9
			1.104	1.062
			1.104	1.062
		2	1.053	1.011	1.033
			1.091	1.049
			1.080	1.038
		3	1.116	1.074	1.071
			1.118	1.076
			1.105	1.063
Positive Control (8N KOH)	60 Minutes	1	0.044	0.001	0.001	0.001	0.1	0.10	0.0	0.0	Corrosive
			0.045	0.002
			0.044	0.001
		2	0.044	0.001	0.001		0.1
			0.045	0.002
			0.044	0.001
		3	0.045	0.002	0.001		0.1
			0.043	0.000
			0.043	0.000

Treatment	Exposure Time	Tissue Replicate	O.D.	Corrected O.D.	Mean of Corrected O.D.	Mean O.D. of Replicate tissues	% Viability/ Tissue	Mean % Viability	SD of % Viability	% CV of % Viability	Corrosivity Class
Reaction Mass (Fenchene, Laevo Alpha Pinene, Laevo Camphene, Dextro Camphene)	3 Minutes	1	0.870	0.828	0.827	0.827	84.9	84.9	0.75	0.9	Non-corrosive
			0.879	0.837
			0.858	0.816
		2	0.872	0.830	0.820		84.2
			0.849	0.807
			0.866	0.824
		3	0.878	0.836	0.835		85.7
			0.875	0.833
			0.877	0.835
	60 Minutes	1	0.323	0.281	0.28	0.278	27.0	26.8	0.15	0.56
			0.323	0.281
			0.319	0.277
		2	0.324	0.282	0.278		26.8
			0.319	0.277
			0.317	0.275
		3	0.316	0.274	0.277		26.7
			0.316	0.274
			0.324	0.282

Key: O.D. = Optical density, S.D. = Standard deviation, C.V. = Coefficient variation

Note: For Negative control, SD and CV of % viability was calculated using OD at 570 nm and for the test item and positive control SD and CV of % viability was calculated using % viability/tissue

Table 2: Data Summary of Adapted Control for Non-Specific MTT Reduction (NSMTT)

Treatment	Exposure Time	Tissue Replicate	O.D.	Corrected O.D.	Mean of Corrected O.D.	Mean OD of Replicate tissues	% NSMTT	Mean % NSMTT	TODTT	% Relative Viability
Negative Control (Untreated killed tissues)	60 minutes	1	0.264	0.221	0.231	0.233	NA	NA	NA	NA
			0.275	0.232
			0.282	0.239
		2	0.277	0.234	0.234
			0.277	0.234
			0.277	0.234
Positive Control (8N KOH)	60 minutes	1	0.047	0.004	0.004	0.004	-22.1	-22.1	NA	NA
			0.047	0.004
			0.047	0.004
		2	0.047	0.004	0.004		-22.1
			0.047	0.004
			0.047	0.004

Keys: O.D. = Optical Density, NSMTT = Non-specific MTT reduction calculation, NA = Not applicable, TODTT = Treated tissue true metabolic conversion,

Note: 1) For positive control (exposure period of 60 minutes) NSMTT was < 0% relative to the negative control, hence true MTT metabolic conversion of treated tissue was not determined; 2) Mean ODNC for 60 minutes exposure was 1.037; 3) Distilled water was used for Negative control tissues and 8N KOH was used for positive control tissues

Interpretation of results:

other: No category (CLP Regulation EC no. 1272/2008)

Conclusions:

Under RHE test method performed in SkinEthic RHE® model the test item does not have to be classified as skin corrosive (cat. 1).

Executive summary:

An in vitro skin corrosion test for the test item was performed in a reconstructed human epidermis SkinEthic RHE® model, according to OECD TG 431 (GLP study). Three epidermis units were treated with 40 µL test item for 3 minutes at room temperature and for 1 hour, at 37°C, 5% CO2. Exposure of the test item was terminated by rinsing with 20 x 1 mL of DPBS. The viability of each disk was assessed by incubating the tissues with MTT, extracting the precipitated formazan crystals using isopropanol, then incubating the tissues for overnight at room temperature, protected from light, and finally measuring the concentration of formazan by determining the OD at 570 nm. Under the test conditions, the mean percent viabilities of the treated tissues were 84.9% (for 3 min exposure) and 26.8% (for 60 min exposure) versus 0.1% (for 60 min exposure) of the positive control item (potassium hydroxide 8N). Therefore, the test item must be considered as non-corrosive.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

11 November 2017 - 12 November 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: Deonar Abattoir slaughter house, Mumbai, Maharashtra.
- Characteristics of donor animals (e.g. age, sex, weight): 1-5 years of age.
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): transported under cold condition in Hank's Balanced Salt Solution containing antibiotics (penicillin at 100 IU/mL and streptomycin at 100 μg/mL).
- Time interval prior to initiating testing: eyes were used within 24 h from the slaughtering.
- indication of any existing defects or lesions in ocular tissue samples: Eyes were examined prior to use. Corneas from eyes free of visible defects were used.
- Indication of any antibiotics used: yes, penicillin at 100 IU/mL and streptomycin at 100 μg/mL.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 750 μL
- Concentration (if solution): undiluted

Duration of treatment / exposure:

10 min ± 30 s

Duration of post- treatment incubation (in vitro):

2 h ± 10 min

Number of animals or in vitro replicates:

3 replicates.

Details on study design:

SELECTION AND PREPARATION OF CORNEAS: All eyes were examined prior to use, corneas free from defects were dissected to a 2-3 mm rim and transferred to a container with Hank's Balanced Salt Solution. Corneas from eyes free of visible defects and with an opacity value inferior than 7 were used. The selected corneas were mounted on the corneal holders with the endothelial side against the O-ring of the posterior half of the holder. The anterior half of the holder was then placed on top of the cornea and fixed with screws. Both chambers were filled with pre-warmed phenol red free Eagle's Minimum Essential Medium (EMEM) and equilibrated at 32 ± 1ºC for at least 1h. Following the equilibration period, the medium was removed from both chambers and baseline opacity readings were taken for each cornea.

QUALITY CHECK OF THE ISOLATED CORNEAS: Corneas from eyes free of visible defects and with an opacity value inferior than 7 were used.

NUMBER OF REPLICATES: 3

NEGATIVE CONTROL USED: yes. Normal saline (0.9% w/v, Sodium Chloride Injection I.P.), source: Realcade Life Sciences Pvt. Ltd., batch no: R2010266.

POSITIVE CONTROL USED: yes. N,N-Dimethylformamide, source: Sigma Aldrich, batch no: BCBQ8511V.

APPLICATION DOSE AND EXPOSURE TIME: 750 μL of undiluted test item, 10 min exposure.

TREATMENT METHOD: closed chamber.

POST-INCUBATION PERIOD: no.

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: the corneal epithelium was washed until no visual evidence of test item was observed using EMEM (containing phenol red) and, once the medium was free of test item, a final rinse with phenol red-free EMEM was performed.
- POST-EXPOSURE INCUBATION: After rinsing, the corneas were incubated for an additional period of approximately 2 hours ± 10 minutes at 32 ± 1ºC.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: corneal opacity was measured with an opacitometer BASF-OP3.0 (Duratec, Germany)
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of microtiter plate reader (OD490)

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA: as indicated in the TG (see table below).

Irritation parameter:

in vitro irritation score

Run / experiment:

mean

Value:

1.2

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

0.94

Positive controls validity:

valid

Remarks:

140.10

Irritation parameter:

cornea opacity score

Run / experiment:

mean

Value:

0.91

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

0.67

Positive controls validity:

valid

Remarks:

106.18

Irritation parameter:

other: permeability

Run / experiment:

mean

Value:

0.019

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

0.018

Positive controls validity:

valid

Remarks:

2.261

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: no.

DEMONSTRATION OF TECHNICAL PROFICIENCY: yes

ACCEPTANCE OF RESULTS:
- A test is considered acceptable if: the positive control gives an IVIS that falls within two standard deviations of the current historical mean and negative/solvent controls gives values of opacity and permeability lower than upper limits for background values.
- Acceptance criteria met for negative control: yes (negative: opacity upper limit=2.52, permeability upper limit=0.099)
- Acceptance criteria met for positive control: yes (historical value range: 63.28 - 257.23, SD: 56.79; mean = 159.82, 2*SD = 113.57)

Table 1. In vitro Irritation Score.

Normal Saline (0.75 mL)
Cornea Holder N°	Io  (LUX)	I (Initial) (LUX)	Initial Opacity Value	I (Post Treatment) (LUX)	Post Treatment Opacity Value	Corr. Opacity Value	OD490 Value	Corr. OD490 Value	IVIS
14	1111	1010	4.41	984	5.56	1.15	0.063	0.018	1.42
15	1086	974	5.00	946	6.32	1.32	0.064	0.019	1.61
16	1105	980	5.50	990	5.05	-0.45	0.061	0.016	-0.21
Mean						0.67	-	0.018	0.94
SD						0.98	-	0.002	1.00

N,N-Dimethylformamide (0.75 mL)
Cornea holder N°	Io  (LUX)	I (Initial) (LUX)	Initial Opacity Value	I (Post Treatment) (LUX)	Post Treatment Opacity Value	Corr. Opacity Value	Final Opacity Value	OD490 Value	Corr. OD490 Value	Final OD490 Value	IVIS
11	1118	998	5.21	289	114.71	109.50	108.83	2.074	2.029	2.011	139.00
12	1114	996	5.14	297	110.02	104.88	104.21	1.464	1.419	1.401	125.23
13	1097	981	5.13	290	111.29	106.16	105.49	3.434	3.389	3.371	156.06
Mean							106.18	-	2.279	2.261	140.10
SD							2.39	-	1.009	1.009	15.44

Reaction Mass (Fenchene, Laevo Alpha Pinene, Laevo Camphene, Dextro Camphene) (0.75 mL)
Cornea holder N°	Io  (LUX)	I (Initial) (LUX)	Initial Opacity Value	I (Post Treatment) (LUX)	Post Treatment Opacity	Corr. Opacity	Final Opacity	OD490	Corr. OD490	Final OD490	IVIS Score
17	1088	970	5.27	934	6.99	1.72	1.05	0.099	0.054	0.036	1.59
18	1047	938	5.05	891	7.40	2.35	1.68	0.076	0.031	0.013	1.88
19	1074	952	5.53	938	6.20	0.67	0.00	0.072	0.027	0.009	0.14
Mean							0.91	-	0.037	0.019	1.20
SD							0.85	-	0.015	0.015	0.93

Keys: IVIS = In Vitro Irritation Score, Io =Baseline Reading (With medium but without cornea), I = LUX.Reading with Medium and Cornea, OD₄₉₀= Optical Density at 490 Wave Length, - = Not Applicable, Corr. = Corrected. Blank OD₄₉₀value = 0.045.

- InitialOpacity Value = [((Io⁄I)-b)/a)], Where I = Initial LUX, Io = Baseline Reading, Note: a (0.0251) and b (0.9894) are constant.

- Post TreatmentOpacity Value = [((Io⁄I)-b)/a)], Where, I = Post Treatment LUX, Io = Baseline Reading.

- Corr. Opacity Value = Post Treatment Opacity Value - Initial Opacity Value

- Final Opacity Value = Corr. Opacity Value – Mean Corr. Opacity Value of Control (Group I)

- Corr. OD₄₉₀Value = OD₄₉₀Value – Blank OD₄₉₀Value, Where Blank Value for this trial was 0.045

- Final OD₄₉₀Value = Corr. OD₄₉₀Value –Mean Corr.OD₄₉₀Value of Control (Group I)

- IVIS (Control) = Corr. Opacity Value + (15 x Corrected OD₄₉₀)

- IVIS (Treatment) = Final Opacity Value + (15 x Final OD₄₉₀)

Interpretation of results:

other: Not classified (CLP Regulation EC no. 1272/2008)

Conclusions:

The test item is not irritating to the eye (no category) as the mean IVIS score in the BCOP test was found to be 1.20.

Executive summary:

An in vitro Bovine Corneal Opacity and Permeability study was conducted in order to determine the potential severe eye damaging effects of the test item according to the OECD guideline 437 under GLP conditions. Three sets consisting of three corneas each were tested: the first set was the negative control, and was treated with 750 μL normal saline; the second set was the positive control, and was treated with 750 μL of dimethylformamide and the third set was treated with 750 μL of test item. The corneas were exposured for 10 min after which the test item was washed and then the corneas were kept in incubation for 2 h at 32ºC. After incubation, opacity of the corneas was measured. Then, to determine permeability, 1 mL fluorescein solution (4 mg/mL) was applied on the anterior surface of the corneas, while fresh EMEM (phenol red-free) was added to the posterior chamber and, after 90 min incubation at 32ºC, the OD (490 nm) of the medium in the posterior chamber was measured. Acceptance criteria were met for positive and negative controls. The mean corneal opacity for the test item treated corneas was 0.91, the mean permeability was 0.0019 and the mean IVIS score was found to be 1.20. Therefore, the test item is not irritating to the eye (no category).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin irritation. Key study. An in vitro skin irritation test of the test item was performed in a reconstructed humanSkinEthic RHE®model, according to OECD TG 439 (GLP study). Three epidermis units were treated with 16μL test item for 42 minutes at room temperature. Exposure of the test item was terminated by rinsing with 25 x 1 mL of DPBS. The epidermis units were then incubated at 37°C for 42 hours in an incubator with 5% CO2. The viability of each disk was assessed by incubating the tissues with MTT, extracting the precipitated formazan crystals using isopropanol and incubating the tissues for overnight in a refrigerator protected from light, and finally measuring the concentration of formazan by determining the OD at 570 nm. Under the test conditions, the mean percent viability of the treated tissues was 2.4%, versus 1.2% of the positive control (5% Sodium Dodecyl Sulfate) and 100% of the negative control (DPBS). Therefore, the test item must be considered as classified for skin irritation (Cat. 2).

Skin corrosion. Key study. An in vitro skin corrosion test for the test item was performed in a reconstructed human epidermis SkinEthic RHE® model, according to OECD TG 431 (GLP study). Three epidermis units were treated with 40 µL test item for 3 minutes at room temperature and for 1 hour, at 37°C, 5% CO2. Exposure of the test item was terminated by rinsing with 20 x 1 mL of DPBS. The viability of each disk was assessed by incubating the tissues with MTT, extracting the precipitated formazan crystals using isopropanol, then incubating the tissues for overnight at room temperature, protected from light, and finally measuring the concentration of formazan by determining the OD at 570 nm. Under the test conditions, the mean percent viabilities of the treated tissues were 84.9% (for 3 min exposure) and 26.8% (for 60 min exposure) versus 0.1% (for 60 min exposure) of the positive control item (potassium hydroxide 8N). Therefore, the test item must be considered as non-corrosive.

Eye irritation. Key study. An in vitro Bovine Corneal Opacity and Permeability study was conducted in order to determine the potential severe eye damaging effects of the test item according to the OECD guideline 437 under GLP conditions. Three sets consisting of three corneas each were tested: the first set was the negative control, and was treated with 750 μL normal saline; the second set was the positive control, and was treated with 750 μL of dimethylformamide and the third set was treated with 750 μL of test item. The corneas were exposured for 10 min after which the test item was washed and then the corneas were kept in incubation for 2 h at 32ºC. After incubation, opacity of the corneas was measured. Then, to determine permeability, 1 mL fluorescein solution (4 mg/mL) was applied on the anterior surface of the corneas, while fresh EMEM (phenol red-free) was added to the posterior chamber and, after 90 min incubation at 32ºC, the OD (490 nm) of the medium in the posterior chamber was measured. Acceptance criteria were met for positive and negative controls. The mean corneal opacity for the test item treated corneas was 0.91, the mean permeability was 0.0019 and the mean IVIS score was found to be 1.20. Therefore, the test item is not irritating to the eye (no category).

Justification for classification or non-classification

Based on the available information, the test item is classified as skin irritation (category 2) in accordance with CLP Regulation (EU) No. 1272/2008.

Based on the available information, the test item is not classified for serious eye damage/eye irritation in accordance with CLP Regulation (EU) No. 1272/2008.